Field of the Invention
The present invention relates to the biomedical field, and particularly relates to a novel Hsp90 inhibitor and a preparation method and use thereof.
Description of Related Art
Heat shock proteins (Hsps) are a class of proteins produced by cells under some stress conditions, such as heat shock, glucose starvation, or pathogenic microorganism infection. They are also widely distributed in normal state cells. Hsps are a family of highly conserved proteins expressed in organisms during evolution, which, as molecular chaperones, are involved in the protection of cells against various stimuli (including cold, hot, anoxia, heavy metal ion, virus infection, etc.). Hsps mainly are involved in folding and transporting nascent peptide chains in cells, and identifying denatured proteins, thus playing a role in regulating cell growth, differentiation, and survival. Hsps are not only involved in the critical physiology processes, such as stress protection, signal transduction, immune response, development, and differentiation, etc., but also relate to the formation of various diseases, such as infection, autoimmune disease, atherosclerosis, tumor, etc.
Based on their molecular weight, Hsps can be divided into 5 large families, i.e., Hsp100s, Hsp90s, Hsp70s, Hsp60s, and small Hsps. Hsp90s are one of the most active molecular chaperones in cells, and are widely involved in signal transduction, hormone response, and transcription regulation processes in cells. The main functions of Hsp90s are maintaining protein stability in cells, improving the resistance of cells against stresses, enhancing antioxidation reactions, and maintaining the normal physiologic function of cells. Hsp90s, themselves, do not involve in the constitution of the target protein. Hsp90s are cytoplasmic proteins, and in mammalian cells, the Hsp90 family consists of 3 members: cytoplasmic chaperone Hsp90-a (inducible type/major type) and Hsp90-b (constitutive type/minor type), analogue endoplasmic reticulum chaperon GRP94 (glucose-related protein 94), and mitochondrial homolog Hsp75/TRAP1 (tumor necrosis factor receptor-associated protein 1). When a stress occurs, Hsp90s can interact with those proteins whose conformations are changed due to the environment stimuli, so as to ensure the proper folding of the proteins and prevent them from the nonspecific aggregation, thus maintaining the normal activity of the cell. Before transmembrane transporting, the proteins must be unfolded, and after transmembrane transporting, they will be refolded to form mature types. Hsp90s, as molecular chaperones, have the unfoldase function, which can identify and bind hydrophobic surfaces that are partially exposed after a proteins unfolds, so as to prevent the interaction from agglomeration, until the transmembrane delivery is finished.
Abnormal activation and mutation proliferation signal molecules that exist in tumor cells have to combine with Hsp90s to stabilize their structure, thus maintaining the growth advantageous properties of tumor cells. In tumor cells, Hsp90s are mainly in the activated state, and in normal cells, they are mainly in the latent state. When in the activated state, Hsp90s can form complexes with the receptor proteins and auxiliary molecular chaperones, Hsp70, Hsp40, Hop (Hsp70 organizing protein), p23, CDC37, etc., thus maintaining the receptor proteins in the mature functional conformations, and protecting the receptor proteins from degradation by proteasomes.
In the normal state, Hsp90s are expressed at low levels, are regulated by the cell cycle, and mainly exist in the cytoplasm. When a stress occurs, Hsp90s rapidly enter the cell nucleus, and induced synthesis of Hsp90s is up-regulated at both the transcription and translation levels, which can increase the anti-stress ability of the cell. However, in tumor cells, Hsp90s show a sustaining high induced expression. Such high expression does not require thermal stimulus, mutation, or abnormal protein, which are known to stimulate the synthesis of Hsp90s. The high levels of Hsp90s is one of the reasons for tumor cells to be hypersensitive to Hsp90 inhibitors.
In 1999, Klein first proposed a tumor multi-point-attack (Multi-enzyme-targeted) theory. The core of such a theory was to use a target site to achieve a multiple point block on the tumor signal pathway network, thus completely destroying the whole signal pathway network on which the tumor depended for survival. Therefore, finding the effective target was a key for applying the tumor therapy. The inhibitor based on molecular chaperones did not directly act on the kinase itself, but inhibited the associated molecular chaperone which maintained the active conformation of the kinase, through the ubiquitin-proteasome pathway, a large amount of kinases were degraded by proteasomes, thus deactivating the signal transduction pathway mediated by the kinases and failing to receive the signal from upstream. As compared with the direct inhibitor of conventional kinases, the inhibition of a single target of Hsp90s can simultaneously produce the feature of multipath antitumor effects, which not only can use a single medicament but also can reduce the drug resistance occurrence, thus making Hsp90s the exciting molecular targets for tumor therapy.
In addition, Hsps also have an intimate relationship with virus infections. At present, it is proved that Hsps induced by a virus after infecting the host, can combine with the virus protein so as to form complexes. Such a process can be associated with virus replication. The Hsps produced by the host cell which are induced by various types of virus infection can combine with the virus protein in a plurality of links during virus replication, forming HSP-virus peptide complexes, facilitating correct folding of the virus protein, facilitating transmembrane transport and virus assembly, maturing of the virus, etc. Thus, it is advantageous for virus replication.
It was reported that herpes simplex virus can induce an Hsp70 of 70 Kda, which required the synthesis of early virus protein, but did not require the replication of the virus DNAs. Hsp70 was expressed at a low level in non-stress rodent cells, but HSV infection can induce its expression. Within 4 hours after the HSV-1 and HSV-2 infections, the levels were increased. The Hsp70 synthesis and accumulation were increased in the infected cells, the UV irradiated HSV-1 lost the activity for inducing Hsp70, and the inhibition on virus DNAs did not affect the induction to Hsp70, but the protein synthesis within 2 hours after infection was necessary for inducing Hsp70.
Herpes simplex virus, which belongs to herpes virus family, and a herpes virus subfamily, was the first discovered human herpes virus, and was divided into two serotypes, i.e., type I (HSV-1) and type II (HSV-2). The herpes simplex virus infection is quite widespread, which mainly infringes skin, mucous membranes, and nervous tissue, thus causing the infection in human and many animals. HSV-1 generally infects the skin and the mucous membrane of the mouth, lips, eyes, and the central nervous system, occasionally the genital organ; HSV-2 generally relates to the genital organ infection and the neonatal infection.
At present, due to the lack of an efficient virus vaccine, drug therapy has become a main approach for treating HSV infection. In the clinic, the common therapeutic drugs are mainly nucleosides such as acycloguanosine, etc., whose target is the viral DNA polymerase, which in turn effects the replication of the virus. At present, the widely used ones are acycloguanosine analogues, with Acyclovir (ACV) as a representative, comprising Acyclovir, Valaciclovir, Penciclovir, Famciclovir and Ganciclovir. In addition, there are also nonnucleoside analogues, with sodium pyrophosphate as a representative. But part of the nucleoside analogues, such as Iododeoxyuridine, trifluorothymidine, arabinosyladenosine, and Ganciclovir, and the like, has mutagenicity, and low safety. And in the 1980s, an ACV resistant strain was discovered, and it was determined that in bone marrow transplantation patients and HIV patients, drug resistant strains were more likely to occur. Therefore, there is a need to find an antivirus drug with a new mechanism of action, and due to the critical role of Hsp90 in virus infection, it also becomes a potential antivirus target.
The first Hsp90 inhibitor drug, geldanamycin (GA), is a benzoquinone drug which was originally screened as an anti-fungal agent. GA is a specific Hsp90 inhibitor. Its structure is mainly formed by connecting a benzoquinone part and a planar macrocyclic Ansa bridge. One study revealed that its antitumor ability depended on the degradation of the tumorigenic protein kinase in the proteasome, but GA has high toxicity to kidney and liver, which may be attributed to its off-target effect. During screening low toxic derivatives at the National Cancer Institute (NCI), and it was found that 17-allylaminogeldanamycin (17-AAG) wherein one side chain of GA was replaced, had all of the characteristics of GA, including the inhibition effect and the antitumor activity of Hsp90, but had lower toxicity. However, 17-AAG had poor water solubility, and cannot be applied orally. Subsequently, a novel derivative of GA (17-dimethylaminoethylamino-17-demethoxygeldanamycin, 17-DMAG) was developed. This compound presented good water solubility and oral bioavailability, and has entered Phase II/III clinic trials for solid tumor and hematologic malignancy.
Radicicol, a macrocyclic antibiotic separated from Monosporium Bonorde, has the potential to reverse malignant phenotypes, which is similar to GA, and plays a role in the Hsp90 receptor protein degradation. Radicicol can reverse the deterioration degree of fibrous cells transfected with v-src and v-Ha-Ras. Radicicol binds to the N-terminal area of Hsp90, and has good antitumor activity in vitro, but has no antitumor ability in vivo, which is mainly attributed to its chemical structure being unstable, and prone to degradation after entering the body. Recently, studies have shown that Novobiocin can significantly decrease the levels of p185erb2, p60v-src, Raf-1, and mutant p53 in cells. It has also been demonstrated in a point mutation experiment that Novobiocin is a C-terminal inhibitor of Hsp90. The studies of Neckers et al., showed that 3 Coumermycins compounds (Novobiocin, Chlorobiocin, Coumermycin A1) all significantly decreased the levels of P185erb2, p60v-src, hypoxia inducible factor 1, and mutant P53 in cells. Novobiocin is a Coumermycin antibiotic which has been used in the clinic and has little toxicity and has good pharmacokinetic characteristics. Novobiocin is a C-terminal inhibitor of Hsp90, and has an inhibitory effect on various of cancer cells. It can be applied in combination with an anticancer drug to reverse the drug resistance to the anticancer drug, but the concentration of Novobiocin used as an inhibitor of Hsp90 is up to 700 μM, which also limits its further development as an antitumor drug in vivo.
In addition to the several Hsp90 inhibitors described above, recently some Hsp90 inhibitors have been synthesized and screened successively. PU3, a purine-scaffold Hsp90 inhibitor, is a micromolecular compound designed based on the result from X-ray crystal diffraction. The action site of PU3 is consistent with that of GA, which acts on the ATP/ADP binding site at the N-terminal of Hsp90, and PU3 is also similar to GA in terms of the inhibition of Hsp90 receptor protein degradation and antitumor ability. PU3 is modified and improved so as to form a derivative thereof, which binds the N-terminal of Hsp90, with an affinity of 30 times higher than that of PU3, but its activity is less than that of 17-AAG. However, the derivative does not present a specific accumulation between cells, but such a characteristic is typical for the more hydrophobic GA derivative. IPI-504 (17-AAG hydroquinone), is mainly aimed at the multiple myeloma treatment, which currently has entered Phase I clinic trials. The compound has high water solubility and can be converted into the active form of 17-AAG in the body. However, the anticancer mechanism thereof is still under research now. NVP-AUY922 is an isoxazole derivative, which also belongs to a micromolecular Hsp90 inhibitor, and has a potential therapeutic effect on the ER- and ERBB2-positive breast cancer patients.